User‐demand fast‐curable ocular glues enforced by multilength tunable networks

Abstract Achieving fast and secure wound closure without ocular foreign body sensation is highly desired in ophthalmologic surgery. Sutureless approaches using tissue adhesives are gaining popularity, but their practical use is limited by the difficulty in controlling adhesion time and satisfying safety standards without compromising adhesive performance. Herein, we report user‐demand hydrogel‐forming ocular glues based on multilength photo‐crosslinkable hyaluronic acid (HA), achieving firm tissue adhesion under wet and dynamic conditions and possessing cornea‐like optical transparency. The HA‐based photocurable glue (HA photoglue) quickly seals wounds upon nontoxic low‐energy light exposure (320–500 nm, < 5 s, < 1 J cm−2), and its mechanical and adhesive properties are improved by introducing short and long crosslinkable moieties into HA through one‐step synthesis, forming multilength networks. Furthermore, the HA photoglue provides stable sealing in wet environments like ocular mucous surface, a clear vision with a light transmittance of more than 95% over the entire visible range, and a lubricating surface with minimal ocular sensation (generating less than 10% frictional force than suture groups). In a rabbit corneal incision model, the HA photoglue showed improved wound healing efficacy based on histological evaluation compared to control groups.


| INTRODUCTION
Corneal injuries associated with anterior fluid outflow require an immediate watertight seal to reduce the risk of infection and leak-related complications, such as hypotony, iris prolapse, corneal swelling, and lens dislocation. 1,2 Although clear corneal incisions inducing self-sealing are commonly used in cataract surgeries (over 23 million surgeries annually worldwide), fast and reliable incision closure is necessary to reduce the potential risk of postoperative infections and improve postoperative visual acuity. 3,4 Ophthalmic suturing has been used as the gold standard for incision closure, but it is delicate and time-consuming procedure that requires good technical skills. In addition, the sutured sites may lead to potential drawbacks, including leakage, secondary damage of ocular tissue, and infection. 5,6 Recently, sutureless approaches using glue-type tissue adhesives have gained popularity as an alternative to corneal sealing and repair. 7 Depending on the polymerization (or gelation) mechanism of liquid formulations, tissue glue can be classified into two types: physical or chemical adhesives. 8 Physical adhesives involving polymer gelation by physical interactions, such as hydrogen bonding, ionic association, and molecular entanglements, form noncovalent and reversible crosslinks within the polymer network. 9 Thus, they often exhibit low mechanical properties (break when the ocular blinking is applied), poor adhesive performance on wet tissues, and long gelation times. To secure a firm and reliable tissue adhesion, chemical adhesives based on reactive chemical polymerization or light-triggered radical polymerization (or photo-crosslinking) are mainly used in wound closure, including corneal sealing. For example, cyanoacrylate glue is widely used for ocular injuries and defects, due to its instant strong adhesion to a wet corneal surface. 10 However, several complications associated with low biocompatibility, poor transparency, and difficulty in applying the glue to a target location due to rapid polymerization upon contact with any fluid have been reported. 4,11 Recently, in situ hydrogel-forming adhesives (ReSure ® ) using reactive macromers (such as N-hydroxylsuccinimidefunctionalized polyethylene glycol) and crosslinkers (such as trilysine acetate) were approved by the U.S. Food and Drug Administration (FDA) as ocular sealants to prevent leakage following cataract surgery with intraocular lens placement. 12 This hydrogel adhesive is beneficial for providing a soft and lubricious surface barrier with increased biocompatibility; however, the uncontrollable gelation that is initiated immediately after mixing the reactive macromers and crosslinkers limit its use. These chemical tissue adhesives formed by reactive covalent crosslinking reactions are challenging to achieve user demands with respect to tissue adhesion and mechanical properties suitable for dynamic ocular environments (e.g., blinking).
Alternatively, hydrogel-forming photo-crosslinkable tissue adhesives composed of hydrophilic polymers functionalized with radical-sensitive groups (namely, acrylate or methacrylate groups) and radical-generating photoinitiators (PIs) have been gaining attention owing to their broad controllability in mechanical properties, curing kinetics, and adhesive performance depending on the crosslinking density. In addition, naturally derived biopolymers functionalized with photo-crosslinkable groups are beneficial for wound healing after forming three-dimensional hydrogel networks by photo-crosslinking at the wound sites. 4 For instance, methacrylate (MA)-functionalized gelatin (GelMA)-based tissue glue exhibited good adhesive and regenerative performance in a corneal injury model. 13 However, GelMA hydrogel adhesives normally require long light exposure and high PI concentrations that could induce cytotoxicity during or after photo-crosslinking, 14 thus, potentially limiting their use in clinical applications. Although other natural polymers, including hyaluronic acid (HA), alginate, and chitosan, have also been used for ocular applications by the introduction of photo-crosslinkable moieties (e.g., MA) to polymer chains, 13,[15][16][17][18][19][20][21] photocuring conditions requiring long exposure to highenergy light sources exceed the general guidelines for limiting light exposure to the eye (1 J cm À2 in 315-400 nm). 22 In addition, the low substitution rate of photosensitive groups in hydrogel-forming photocurable glues may result in poor tissue adhesion due to low mechanical strength and vulnerability to external stress. 23 Tough adhesive hydrogels with multiscale networks showed good mechanical stability under applied force by the energy dissipation mechanism. 24 Generally, multi-network or double-network hydrogels have been prepared through additional polymerization or crosslinking in existing hydrogel network. 25,26 One of the hydrogel networks provides material strength due to its tightly crosslinked structure, while the other loosely crosslinked structure could dissipate energy across the network through its flexible moieties. However, multiple preparation steps to form the tough adhesive limit their application in clinics, which require rapid application in emergency situations.
A suitable approach to overcome the limitations of existing ocular adhesives should (i) offer strong tissue adhesion on wet and dynamic mucosal surfaces, (ii) enable fast curing while meeting safety guidelines of light exposure to the eye, (iii) have proper mechanical properties withstanding cyclic external stress, (iv) be amendable to quick application (user-demand adhesion), (v) provide soft and lubricous surface to minimize the ocular foreign sensation, (vi) be transparent without loss of visibility, and (vii) be biocompatible to promote wound healing process.
Here, we report a highly engineered photocurable ocular glue that provides a multilength-networked, transparent, watertight hydrogel barrier on the wound site following low-energy light exposure (less than 1 J cm À2 ). Hydrogel-forming fast-curable glue is designed to form a multilength network structure based on copolymeric HAs with short and long photo-crosslinkable groups, enabling increased mechanical stability and tissue adherence during the wound healing process ( Figure 1). By controlling the substitution ratio of short and relatively long photo-crosslinkable moieties in HA polymer chains through onestep synthesis, the mechanical and adhesive properties of the photocured hydrogels were optimized to improve their flexibility without decreasing the mechanical strength. The formulation of HA-based photocurable glue (HA photoglue) consisting of photo-crosslinkable HAs and PIs has been determined to improve its usability and safety for easy clinical application, without affecting its fast-curing performance (less than 5 s). The long-term stability and lubricity of HA photoglues under wet and dynamic conditions were evaluated through in vitro burst tests and ex vivo friction tests, respectively. The HA photoglue containing approximately 90% water after photo-crosslinking showed excellent transparency with a light transmittance of more than 95% over the entire visible range. Finally, the wound healing efficacy and biological safety of the HA photoglue were assessed using a rabbit corneal incision model.

| Synthesis of photo-crosslinkable polymers
To synthesize HA-based photo-crosslinkable polymers, HA (1.0 g) was dissolved in a mixture of 10 ml distilled (DI) water and 2 ml dimethyl sulfoxide. The HA solution was cooled to 5 C and the pH was adjusted to 8.0 using a 1 M NaOH solution. A mixture of five equivalents of MAA and PEA (4:1, 3.5:1.5, or 2.5:2.5) with respect to the disaccharide unit of HA was added dropwise over a period of 1 h.
The pH was simultaneously maintained between 8.0 and 10.0 by adding 1 M NaOH solution. The temperature and pH were maintained for another 23 h, after which the reaction mixture was added slowly to a 94% ethanol and acetone mixture. The obtained precipitate was filtered and washed with 94% ethanol to give copolymeric HA with two photo-crosslinkable groups (HAMA-PA) as a white solid, which was stored at À20 C until use. Methacrylated HA (HAMA), used as a control, was prepared following a method reported previously. 23 Briefly, 1 g of HA was dissolved in 10 ml of DI water and the pH was adjusted to 8.0, using 1 M NaOH at 5 C. Then, four equivalents of MAA, with respect to the disaccharide unit of HA, were added dropwise over a period of 30 min. The pH was simultaneously maintained between 8.0 and 10.0 by adding 1 M NaOH for another 23 h. The reaction mixture was then precipitated in 94% ethanol, and the solid was washed with 94% ethanol, frozen at À30 C, lyophilized, and stored at À20 C until use.
GelMA, used as a control, was prepared following a method reported previously. 23 Briefly, 1.0 g of type A porcine skin gelatin was dissolved in 10 ml phosphate-buffered saline (PBS) at 60 C and 2.58 g MAA was added, and the solution was stirred further for 4 h. The macromer solution was dialyzed against DI water for 1 week at 40 C with frequent changes in DI water to remove the salts and methacrylic F I G U R E 1 Schematic illustration showing the user-demand hydrogel-forming ocular glues based on copolymeric hyaluronic acids (HAs) with short and longphoto-crosslinkable groups, achieving a firm adhesion on wet corneal surface and possessing cornea-like optical transparency. The HA-based photocurable glue (HA photoglue) offers a multilength-networked, transparent, and watertight hydrogel barrier on the wound site following low-energy light exposure acid. The solution was frozen at À55 C, lyophilized, and stored at À20 C until use. 1 H nuclear magnetic resonance (NMR) spectra were obtained using a Bruker 600-NMR spectrometer (Massachusetts, USA). This was used to confirm the functionalization of the photocrosslinkable methacrylate (MA) or 4-pentenoate (PA) groups to calculate the total degree of substitution (n = 3).

| Rheological measurement
Rheological experiments were performed using a stress-controlled rheometer (MCR 302, Anton Paar, Graz, Austria) equipped with a photocuring system (Omnicure S2000) and a temperature-controlled bath. Radiation with an intensity of~230 mW cm À2 was exposed through a transparent bottom-plate fixture (n = 3). A 20 mm aluminum plate was used for the top geometry. Time-sweep oscillatory tests were performed with plate-plate geometry at ambient temperature (24-25 C), 1 Hz frequency, 0.4 mm gap, and 1% strain. To equilibrate and inhibit photo-crosslinking by dissolved oxygen in the solution, a 1-min delay to onset was processed. 27 The gelation of HA photoglues was determined at the time point when the storage modulus (G') inversed the loss modulus (G''). source was installed at a distance of 5 cm to irradiate the entire area of specimens. The mechanical properties of the photo-crosslinked HA photoglues were measured using a mechanical tester (34sc-1, Instron, Massachusetts, USA) in tensile mode at a strain rate of 1 mm min À1 . 23 The tensile strength and elongation were determined at the maximum point of stress and strain, respectively, in the stress-strain curves. The toughness was calculated from the area below the stress-strain curve until fracture. The elastic modulus was evaluated by obtaining the initial 5% of the slope in the strain-stress curves. Five repeats were examined for each group at room temperature (24-25 C).

| Synchrotron-based transmission small-angle X-ray scattering (TR-SAXS) measurement
TR-SAXS were performed at the 9A U-SAXS beamline of the Pohang Accelerator Laboratory (PAL) in Korea. The experimental condition was set to a wavelength of 0.626 Å and sample-to-detector distance of 6.5 m. To avoid interference by X-ray exposure to photocrosslinking in the hydrogel, fully crosslinked HA-based hydrogels were prepared by light exposure for 120 s. The hydrogels of HA photoglues were exposed to an X-ray beam for 10 s under atmospheric conditions without a pre-treatment process. Scattered photons, induced by the homogeneity of HA hydrogels, were collected using a 2D charge-coupled detector (MX170-HS; Rayonix Ltd., Illinois, USA).

| In vitro wound closure test
The adhesive bond strength of the HA photoglues was measured by a wound closure test. 23 Corneal tissue (5 Â 15 mm) was fixed between glass slides leaving a 6 mm section. The tissue was cut apart using a razor blade, and 40 μl of HA photoglues was dropped onto the corneal tissue. After photo-crosslinking, the slides were placed on a mechanical tester for shear testing by tensile loading at a strain rate of 1 mm min À1 . The shear strength was obtained at the maximum stress point. Five repeats were examined for each group at ambient temperature (24-25 C).

| Ex vivo burst pressure test
For the intraocular pressure (IOP) test, a 5-mm corneal incision was created on the explanted porcine cornea (n = 5). HAMA-PA or HAMA solutions (20 μl) were applied at the incision site, followed by curing to seal the incision. Then, an 18-gauge syringe needle connected to a peristaltic pump was inserted into the enucleated porcine eye globes, and PBS was pumped into the sealed porcine eye at a flow rate of 2 ml min À1 until leakage. The IOP was recorded using a water pressure sensor (SMC, Tokyo, Japan).
Burst pressure testing using HA photoglues was conducted using a standard method (ASTM F2392-04) as described in the literature. 13 In brief, a hole was created in the collagen substrate (4 Â 4 cm) using a 3 mm diameter biopsy punch. Then, 20 μl of HA photoglues were dropped on the surface of the hole and the hole was immediately sealed upon light exposure. After fixing the tissue in the measuring apparatus connected to the peristaltic pump, the PBS solution was pumped at a flow rate of 2 ml min À1 until leakage. The peak pressure before the pressure loss was considered as the burst pressure. Five repeats were examined for each group.

| Ex vivo friction test
The friction test to evaluate mechanical sensation by a corneal foreign body was performed after treatment with HA photoglues (20 μl) and sutures (Black silk 7-0, Ailee, Korea) onto the porcine cornea. The HA photoglues and sutures (3 or 7 stitches) were placed at the center of explanted corneal tissue (1 cm Â 1 cm) fixed on a glass slide. The explanted eyelid was fixed on another glass slide and placed over the cornea. The friction force between the treated cornea and eyelid adhered on glass slides was monitored during repetitive movements (100 cycles) that mimicked the blinking eye at room temperature (24-25 C).

| Optical properties of HA photoglues
The transmittance of the photo-crosslinked HA photoglues was measured using a UV-Vis spectrometer (Libra S70; Biochrom, Cambridge, UK) at a wavelength of 200-800 nm. The sample was prepared by filling solutions with a thickness of 500 μm, followed by photocrosslinking as described above. The refractive index (RI) of the HA hydrogels was measured using an ellipsometer (RC2-XF; J.A. Woollam, Nebraska, USA). Three repeats were examined for each group.

| Efficacy test of HA photoglues with in vivo corneal incision model
The   obtained. Remarkably, the total degree of substitution of HA polymer chains for all feed ratios was found to be more than 200%, as confirmed by 1 H NMR analysis, which is suitable for fast photocrosslinking reaction, although the total degree of substitution decreased with the increasing molar concentration of PEA ( Figure S2 and Table S1). This one-step synthetic approach to obtain multilength photo-crosslinkable HA reduces the number of synthetic steps and improves the overall process efficiency, while minimizing time and cost.

| Formulation of a HA photoglues
To determine the composition of HA photoglues for ocular applica-  (Figure 3c, d). The 20% (w/w) polymer solution was not discharged from the syringe at a force below 100 N. The 10% (w/w) HAMA-PA solution was smoothly pressed out in seconds without a high break loose peak, observed in a lower viscous solution (5% (w/w)) ( Figure S4).
As tissue glue is desirable for application in the target area, we

| Rheological analysis and mechanical properties of HA photoglues
The gelation kinetics of HA photoglues were studied through a timesweep oscillatory test with plate-plate geometry. By applying smallamplitude oscillatory shear, the formation of crosslinks and changes in the material properties, such as elastic (G') and viscous (G'') moduli, under light exposure (320-500 nm), were monitored with minimal disruptions to the chemical reaction. 30 The evolution of G' and G" for HA photoglues with different molar ratios of MA and PA was measured under light exposure with 230 mW cm À2 and 1 rad À1 oscillation. As shown in Figure 4a To investigate the mechanical properties of the HA photoglue after photo-crosslinking, hydrogel-type testing specimens were prepared using a dog-bone-shaped mold (Figure 4b). The representative F I G U R E 4 Legend on next page. stress-strain curves of the photo-crosslinked hydrogels of HA photoglues obtained in the tensile tests are shown in Figure 4c. With increasing PA substitution rate in HA, the modulus of the photocrosslinked hydrogels gradually decreased from 350 kPa for HAMA to 50 kPa for HAMA-PA (5:5) (Figure 4d). However, the tensile strength of the hydrogels at fracture did not significantly change with an increase of the PA substitution in the HA polymer chains (Figure 4e).
Interestingly, the HAMA-PA (7:3) hydrogel exhibited increased tensile elongation prior to breaking (60% elongation above the initial length) ( Figure 4f); thus, it showed 110% higher toughness compared to HAMA without PA introduction (Figure 4g). This enhanced resistance against mechanical deformation would be critical to maintain its mechanical integrity under dynamic conditions, such as bending motion in the body (Figures S6 and S7).  (1)).
where λ is the wavelength of the monochromated X-ray beam, and 2θ is the scattering angle.
In Figure 4i, the experimental scattering intensity profiles for all HAMA-PA hydrogels, indicated by scattered symbols, consistently presented the crossover point of the slope change, due to inhomogeneity (or density fluctuation) at the sub-nanometer scale. To extract the microstructural information of the HAMA-PA hydrogels, the scattering intensity (I(q)) profiles were fitted using the correlation length model in Equation (2). 33 where I(0), B, and ξ are the scattering intensities extrapolated to the scattering vector q = 0, background scattering, and correlation length, respectively. All scattering intensity profiles for the HAMA-PA hydrogels were well-fitted to the correlation length model, as indicated by the solid lines in Figure 4i. The correlation length (ξ) of crosslinked domains, extracted from the correlation length model fit, increased with increasing substitution ratio of longer PA groups ( Figure 4i and Table S2). This result suggests that the introduction of photo-crosslinkable PA groups in HA chains manipulates the length between crosslinks (free polymer chains) in the networks. In addition, the power-law exponent n~2 was consistently observed over the entire hydrogel, which is a general value for polymeric gel systems. 34 According to the theory of rubber elasticity for a network of flexible chains, the shear modulus (G') is given by Equation (3)) with respect to ξ. 33,35 where k and T are the Boltzmann constant and absolute temperature, respectively. As shown in Figure 4j approximately 1200 mmHg, which is 80% higher than that of the GelMA group (700 mmHg) (Figure 5d). Even in burst tests using punctured thin collagen sheets (Figure 5e), the HA photoglues showed sufficient burst pressure (~120 mmHg) to withstand normal IOP (20 mmHg) in the human eye. 37 Notably, the burst pressure of the HA photoglues decreased by 10-20% after dipping in PBS for 24 h, highlighting the structural stability of HA photoglues with a high degree of substitution of photo-crosslinkable groups (Figure 5f).
In dissolution tests in a wet environment at 37 C ( Figure S8

| DISCUSSION
The fast-curable hydrogel-forming photoglue based on multilength photo-crosslinkable HAs suggests a new strategy for developing tissue-specific adhesives with high potency for translation into clinics.
By introducing multiple photo-crosslinkable groups of different lengths into HA polymer chains in a controlled manner, the mechanical and adhesive properties can be manipulated by regulating the internal structure of the crosslinked hydrogel network. In particular, the multilength network structure in HA photoglues can be attributed to the effective dissipation of mechanical stress in the short chain, while the relatively longer chain delays the rupture process and consequently increases the elongation. 44 In addition, the single-component glue prefilled in syringes would be advantageous, compared to multicomponent glues using reactive chemical gelation, for fast application without a mixing procedure.
HA is selected as an ideal ocular adhesive material because it is a biodegradable biopolymer present in the vitreous humor of the eye and approved by the FDA for use as a vitreous replacement during eye surgery. 45,46 In addition, HA promotes cell migration and proliferation, thereby facilitating rapid repair of injured corneal wounds. 47,48 Aside from the safety and biological function of HA, it possesses a suitable chemical structure to functionalize effective photocrosslinking. The four OH groups in one repeating unit of HA enable a high degree of substitution of photo-crosslinkable groups, so that the HA photoglue presented in Figure 4 can achieve gelation in 2.5 s under a total light dose of~0.57 J cm À2 .
As shown in Figure 8, compared with previously reported photocuring conditions for tissue adhesives, the HA photoglue requires very short and low light doses (less than 5 s and 1 J cm À2 ) to produce a firm tissue adhesion with good mechanical stability. Given the high molar extinction coefficient (254 M À1 cm À1 at 365 nm) in the UV-Vis region of PI (LAP) used in this study ( Figure S12), it is expected to achieve sufficient adhesion with a lower energy of 0.3 J cm À2 or less if a single wavelength light source of 365 nm is used. Considering the high standard of light safety requirements, this fast and safe photocuring system would be critical for the translation of HA photoglue into the clinic.

| CONCLUSION
In this work, we demonstrated a new tissue glue that provides instant